Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules

Juliana I. Sesma, Silvia M. Kreda, Seiko F. Okada, Catharina van Heusden, Lama Moussa, Lisa C. Jones, Wanda K. O'Neal, Natsuko Togawa, Miki Hiasa, Yoshinori Moriyama, Eduardo R. Lazarowski

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

Original languageEnglish
JournalAmerican Journal of Physiology - Cell Physiology
Volume304
Issue number10
DOIs
Publication statusPublished - May 15 2013

Fingerprint

Mucins
Nucleotides
Adenosine Triphosphate
Organelles
Fluids and Secretions
Purinergic Receptors
Uridine Triphosphate
Adenosine Monophosphate
Adenosine Diphosphate
Small Interfering RNA
Fluorescent Antibody Technique
Western Blotting

Keywords

  • ATP release
  • Mucin granules
  • Mucin secretion
  • SLC17A9
  • VNUT

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Sesma, J. I., Kreda, S. M., Okada, S. F., van Heusden, C., Moussa, L., Jones, L. C., ... Lazarowski, E. R. (2013). Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules. American Journal of Physiology - Cell Physiology, 304(10). https://doi.org/10.1152/ajpcell.00371.2012

Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules. / Sesma, Juliana I.; Kreda, Silvia M.; Okada, Seiko F.; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C.; O'Neal, Wanda K.; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori; Lazarowski, Eduardo R.

In: American Journal of Physiology - Cell Physiology, Vol. 304, No. 10, 15.05.2013.

Research output: Contribution to journalArticle

Sesma, JI, Kreda, SM, Okada, SF, van Heusden, C, Moussa, L, Jones, LC, O'Neal, WK, Togawa, N, Hiasa, M, Moriyama, Y & Lazarowski, ER 2013, 'Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules', American Journal of Physiology - Cell Physiology, vol. 304, no. 10. https://doi.org/10.1152/ajpcell.00371.2012
Sesma, Juliana I. ; Kreda, Silvia M. ; Okada, Seiko F. ; van Heusden, Catharina ; Moussa, Lama ; Jones, Lisa C. ; O'Neal, Wanda K. ; Togawa, Natsuko ; Hiasa, Miki ; Moriyama, Yoshinori ; Lazarowski, Eduardo R. / Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules. In: American Journal of Physiology - Cell Physiology. 2013 ; Vol. 304, No. 10.
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AU - Jones, Lisa C.

AU - O'Neal, Wanda K.

AU - Togawa, Natsuko

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AU - Moriyama, Yoshinori

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N2 - Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

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